Oxaliplatin anti-resistance agent

ABSTRACT

The invention concerns a method for detecting in vitro resistance of cancer cells to an oxaliplatin treatment characterized in that it consists in measuring mitochondrial apoptosis of cancer cells being treated or capable of being treated or to be treated with oxaliplatin.

This invention relates to the treatment of cancer in patients presentingresistance to oxaliplatin.

The invention in particular relates to the diagnosis of resistance ofcolorectal cancers to the anti-tumoral medication “oxaliplatin”(international non-proprietary name of this product, the commercial nameof which is Eloxatine).

The invention also relates to the reduction of this resistance byappropriate treatments using. “anti-resistance” agents, improving theeffectiveness of the oxaliplatin-based treatment (in combination withoxaliplatin or by second intention, after development of oxaliplatinresistance).

Chemotherapeutic treatments of colorectal cancers, in spite of theavailability of active anti-tumoral molecules like oxaliplatin, seetheir efficacy very limited by the frequent occurrence of resistance intumor cells to the cytotoxic effects of the medications, used alone orin combination.

The reduction of this resistance is therefore a major issue for healthcare and the pharmaceutical industry. Anti-cancerous oxaliplatintreatments, the administration of which is aimed at destroying cancercells, are in particular described in documents U.S. Pat. No. 5,716,968and EP 0 943 331.

The attainment of this objective, amounting to the creation of“anti-resistance” treatments combined with anti-tumor medications likeoxaliplatin, requires the identification of molecular mechanisms up tonow not elucidated that govern the emergence of resistance inside tumorcells.

The identification of these mechanisms, unknown at present, of theresistance of cancers, in particular colorectal cancer, to oxaliplatin,therefore aims principally at two applications:

early diagnosis of resistance: it consists of avoiding chemotherapiesthat would have no therapeutic benefit, while they represent a toxicrisk or high cost,

treatment by medications opposing or circumventing the resistancemechanisms.

It is important to note that in prior art there is no early test forresistance to oxaliplatin treatment.

Oxaliplatin resistance: oxaliplatin is a platin salt possessing ananti-tumor activity spectrum much broader than conventional platin saltssuch as cisplatin or carboplatin. The mechanisms of resistance tocisplatin have been for the most part elucidated, but do not takeoxaliplatin resistance into account. More particularly, the deregulationof the MMR or NER repair systems associated with cisplatin resistancedoes not confer resistance to oxaliplatin. Oxaliplatin resistanceremained unexplained until this invention. Oxaliplatin (CgH, 4N204Pt,[(1R, 2R)-1,2-cyclohexanediamine-N,N′] [oxalato (2-)-O,O′] platinum), isa diaminocyclohexane known to damage DNA. This invention coversresistance to oxaliplatin, as well as, should the occasion arise, tooxaplatin derivatives that also give rise to resistance.

Two studies, conducted on the same ovarian cancer cell model (line ATCCA2780), identified a potential effector mechanism for resistance tooxaliplatin in this type of cancer: an increase in intracellularglutathione, as well as a reduction of intracellular accumulation ofplatin and DNA-platin adducts, are associated with resistance tooxaliplatin. But these studies do not provide a functional demonstrationof these identifications. The hypothesis implicating glutathione isemphasized in the document Cancer Lett. 1996 Jul. 19, 105(1):5-14,Altered Glutathione Metabolism in Oxaliplatin Resistant OvarianCarcinoma Cells (Elakawi Z, Abu-hadid M, Perez R, Glavy J, Zdanowicz J,Creaven PJ, Pendyala L.), Department Of Investigational Therapeutics,Roswell Park Cancer Institute, Buffalo, N.Y. 14263, USA.

A hypothesis for the participation of DNA repair mechanisms is advancedin the document Cellular and Molecular Pharmacology of Oxaliplatin, Vol.1, 227-235, January 2002, Molecular Cancer Therapeutic, (Eric Raymond,Sandrine Faivre, Stephen Chaney, Jan Woynarowski and Esteban Cvitkovic).

However, these studies do not make it possible to explain with certaintythe mechanisms of resistance observed.

Thus, the invention aims at mitigating the disadvantages of prior art,and in particular at elucidating the mechanisms of resistance of thecancers, in particular colorectal cancers, to oxaliplatin, in order tobe able to implement early diagnosis of resistance during treatment, andtailor a rational pharmacological approach that can lead to thedevelopment of “anti-resistance” treatments better targeted to thesemechanisms.

Vice-versa, performance of early diagnostic tests for resistance willmake it possible, at least, to inform the oncologist of the necessity ofreorienting treatment (for example by introducing other medications inthe therapeutic scheme). The benefit of this will be the reduction ofadverse effects and the limitation of unnecessary health-careexpenditures. Moreover, the availability of specific treatments(medications, gene therapies, etc.) opposing or circumventing resistanceat the level of demonstrated mechanisms (mitochondrial apoptosis) willrestore the efficacy of oxaliplatin. The benefit will obviously bemedical but also economic: the gain in efficacy will justify themaintenance and extension of oxaliplatin use.

The inventors had to resolve several technical problems including theimplementation of a reliable experimental model (selection andcharacterization of cell lines resistant to oxaliplatin from referencelines) and the exploration of this model (identification of thealteration of mitochondrial apoptosis as marker for specific resistanceto oxaliplatin).

The inventors succeeded in showing that oxaliplatin resistance isassociated with abnormal expression of the mitochondrial apoptosisgenes. Prior art describes apoptosis inducing compounds acting directlyand specifically at the mitochondrial level. However, the connectionbetween mitochondrial apoptosis (MA) and mechanisms of oxaliplatinresistance is not at all described or suggested in prior art.

The inventors have therefore developed a method for the diagnosis ofoxaliplatin resistance, based on the visualization of markers for thealteration of mitochondrial apoptosis in the tumor cells, by anyappropriate means: biochemical such as immunodetection, genetic such assequencing or the quantification of transcripts.

Thus, according to a first characteristic, the invention relates to adetection process, in vitro or in vivo, of the resistance of cancercells to oxaliplatin treatment, comprising the measurement ofmitochondrial apoptosis of cancer cells that are treated or can orshould be treated with oxaliplatin. By resistance of cancer cellstreated with oxaliplatin we mean that the cancer cells, of a patient orin culture, resist oxaliplatin treatment in such a way that thistreatment is not totally satisfactory because it does not make itpossible to destroy them to a sufficient extent.

This detection process relates in particular to colorectal cancers.However, other cancers whose treatment involves administration ofoxaliplatin also belong to the invention, in particular certain cancersof the ovaries, the germinal cells, the lung, the digestive tract, theprostate, the pancreas, the small intestine and the stomach.

According to one realization the detection process involves themeasurement of the expression of at least one gene of mitochondrialapoptosis. By “expression of at least one gene of mitochondrialapoptosis,” we mean the level of expression of at least one effector ormarker gene of mitochondrial apoptosis. By effector gene, we mean a generesponsible at least in part for mitochondrial apoptosis, thisexpression being expressed in particular by the amount of RNA produced,the amount of protein coded by these genes, the level of activity ofthese proteins. For example, a low level of apoptosis can be due to thesynthesis of an apoptosis protein whose sequence differs with respect tothat of a non-resistant patient, the amount of protein being normal butits biological activity being lower. By marker gene we mean a gene thatis not necessarily implicated in the mechanisms of mitochondrialapoptosis, but whose level of expression is correlated with a specifiedlevel of apoptosis.

Among the effector or marker genes for mitochondrial apoptosis, we canin particular analyze, in addition to the genes already studied by theinventors (Bax gene in particular), genes known for their implication inmechanisms of mitochondrial apoptosis, described in particular indocument U.S. Pat. No. 6,268,398:

factors initiating or stimulating the apoptosis cascade and/or theactivity of caspase proteases (Thornberry and Lazebnik, Science281:1312-1316, 1998), such as cytochrome c, which are released followingoxidative stress;

“apoptosis inducing factors” described in Murphy, Drug Dev. Res.46:18-25, 1999;

factors inducing chromatin condensation (Marchetti et al., Cancer Res.56:2033-38, 1996) which precede apoptosis;

Bcl-2 proteins, known for their anti-apoptosis activity, located in theouter mitochondrial membrane (Monaghan et al., J. Histochem. Cytochem.40:1819-25, 1992), which protect the membranes against oxidative stress(Korsmeyer et al., Biochim. Biophys. Act. 1271:63, 1995; Nguyen et al.,J. Biol. Chem. 269:16521-24, 1994) in particular by blocking the releaseof cytochrome c and the activation of caspase 3 (Yang et al., Science275:1129-1132, 1997; Kluck et al., Science 275:1132-1136, 1997).

Those skilled in the art have at their disposal numerous appropriatetechniques for the measurement of gene expression. We cite, for example:

the measurement of mRNA and cDNA, by means of RT-PCR techniques,Northern blot, hybridization to cDNA banks (Sambrook et al., MolecularCloning—A Laboratory Manual, Cold Spring Harbor Press, New York (1989),techniques of differential display (Liang et al., 1995, Curr. Op.Immunol. 7:274-280; EP 534 858), techniques using cDNA probes oroligonucleotides (Eisen, M. B. and P. O. Brown, Methods Enzymol,303:179-205 (1999); Brown, P. O. and D. Botstein, Nat Genet, 21 (1Suppl):33-7 (1999); Cheung, V. G., et al., Nat Genet, 21(1Suppl):15-9(1999));

measurement of proteins by means of western-blot and immunohistochemicalanalyses.

For example, the quantification of cytochrome c can make use of aspectrophotometric or immunochemical method. The release of cytochrome cfrom the mitochondria can be followed, for example, by means ofimmunological methods, by MALDI-TOF spectrometry coupled with affinitycapture (in particular for apocytochrome c and holocytochrome c) and bythe SELDI system (Ciphergen, Palo Alto, USA).

Measurement of the activity of caspases can make use of tests on caspasesubstrates (Ellerby et al., 1997 J Neurosci. 17:6165), such as thelabeled synthetic peptide Z-Tyr-Val-Ala-Asp-AFC, Z being a benzoylcarbonyl group and AFC 7-amino-4-trifluoromethylcoumarin, on nuclearproteins such as UI-70 kDa and DNA-PKcs (Rosen and Casciola-Rosen, 1997,J. Cell. Biochem. 64:50; Cohen, 1997, Biochem. J. 326:1).

To the extent that an abnormally low level of mitochondrial apoptosismay be due to several genes, detection may involve the measurement ofthe level of expression of several genes for apoptosis; it is possibleto determine in this way the profile of expression of several genes thatare compared between patients for whom resistance has been diagnosed andnon-resistant patients. By determining sufficiently precise profiles ofexpression, the clinician can detect a resistant phenotype, and alsopredict resistances in order to optimize the therapy.

The genes for mitochondrial apoptosis may belong to the mitochondrialDNA or to the nuclear DNA.

According to, one realization, the detection process involves themeasurement of the amount of Bax protein in the cancer cells and themeasurement of the mRNAs coding for the Bax protein.

According to one realization the detection process involves:

a) determination of the level of mitochondrial apoptosis and/or of thelevel of expression of at least one gene for mitochondrial apoptosis ofcancer cells sampled from a patient treated with oxaliplatin;

b) comparison of the level of mitochondrial apoptosis with a controlsample from a patient not resistant to oxaliplatin.

A lower level of mitochondrial apoptosis indicates resistance. A lowerlevel of expression indicates resistance in the case of an effector genestimulating mitochondrial apoptosis, a higher level of expressionindicates resistance in the case of an effector gene inhibitingmitochondrial apoptosis.

Deviations in the levels of expression analyzed over a sufficient numberof patients make it possible to determine the risk and the degree ofresistance, the significant quantitative deviations observed being lowor high according to the genes implicated.

It is possible to use samples for example from biopsies on an individualsuffering from cancer at different times. For example, a first samplecorresponds to the time of diagnosis and a second sample is obtained ata second time after treatment of the patient with a compositionconsisting of an anti-resistance agent. The diagnosis can also becarried out following gene therapy, for example to evaluate the level ofmitochondrial apoptosis following the transfer of nucleic acid sequencescoding for the proteins of mitochondrial apoptosis.

The invention also relates to a process for the detection of cancercells resistant to oxaliplatin involving putting the biological sampleexamined together with at least one antibody capable of recognizing anapoptosis protein or a biologically active fragment of this protein, andthe visualization of the antigen-antibody complex that may have formed.

For the implementation of this process a kit can be used consisting of:

a) an antibody that is for example monoclonal or polyclonal, saidantibody being capable of recognizing an apoptosis protein or abiologically active fragment of this protein;

b) possibly reagents for the composition of a medium conducive to theimmunological reaction;

c) possibly reagents making possible the visualization ofantigen-antibody complexes produced by the immunological reaction.

In this way, antibodies can be used to detect under-expressed apoptosisproteins. Most preferably, for a given apoptosis protein, the antibodiesrecognize epitopes of the protein that are not present in otherproteins.

Antibodies designed to specifically recognize one or more epitopes ofapoptosis proteins, in particular the Bax protein, can in particular bemonoclonal, polyclonal, humanized or chimeric antibodies, single chainantibodies, Fab fragments, Fab′2 fragments, fragments produced by an Fabexpression bank and anti-idiotypic antibodies.

Monoclonal antibodies, a homogeneous population of antibodies for aspecific antigen, can be obtained by means of techniques known to thoseskilled in the art, such as the hybridoma technique of Kohler andMilstein (Nature 256:495-497,1975; and U.S. Pat. No. 4,376,110), thetechnique of human B cell hybridomas (Kosbor et al., Immunology Today4:72, 1983; Cole et al., Proc. Natl. Acad. Sci. USA 80:2026-2030, 1983),the technique of EBV hybridomas (Cole et al., “Monoclonal Antibodies andCancer Therapy,” Alan R. Liss, Inc. pp. 77-96, 1985). It is alsopossible to prepare monoclonal antibodies by means of phage display bankkits marketed by Pharmacia or Stratagene.

Chimeric antibodies can be obtained according to a technique of Morrisonet al., Proc. Natl. Acad. Sci., USA 81:6851-6855. Fab expression bankscan be constructed according to the technique of Huse et al., Science246:1275-1281, 1989. Anti-idiotypic antibodies can be obtained by thetechnique of Greenspan and Bona, FASEB J. 7:437-444, 1993.

According to another characteristic, the invention relates to a processfor detection of the resistance of a cancer to oxaliplatin consisting ofthe in vitro or in vivo detection of at least one mutation indicative ofdefective apoptosis of cancer cells in the case of oxaliplatintreatment. The identification of such mutations makes possible earlydiagnosis that makes it possible to better target the therapy and toavoid inappropriate treatments. Comparative sequencing of apoptosisgenes between patients with an early diagnosis of resistance andresistant patients can also be used. Thus the detection process caninclude for example the detection of a mutation in a region of the Baxgene containing a series of 8 deoxyguanines.

The invention also relates to a process for the detection of cancercells resistant to oxaliplatin implementing at least one primer sequenceor specific probe for a mitochondrial apoptosis gene such as the Baxgene, obtained by appropriate techniques of construction using sequencesretrieved for example from GenBank.

The invention thus also relates to a process consisting of:

a)isolation of the mitochondrial DNA from the biological sample to beexamined, or the procurement of a cDNA from the RNA of the biologicalsample or from the genomic DNA;

b) specific amplification of the DNA of a) by means of at least oneprimer for amplification of a mitochondrial apoptosis gene in particularof the Bax gene.

It is thus possible to use a kit for the diagnosis of oxaliplatinresistance consisting of means for extraction of the mitochondrial DNAof cancer cells, means for detection and amplification of mRNA ofmitochondrial apoptosis genes, for example of the Bax gene, or ofgenomic DNA.

The invention also relates to a process consisting of:

a) putting together a nucleotide probe of a mitochondrial apoptosis genesuch as the Bax gene and the biological sample analyzed, the nucleicacid of the sample having, as the case may be, been previously madeaccessible to hybridization, under conditions allowing hybridization ofthe probe and the nucleic acid of the sample,

b) visualization of the hybrid possibly formed.

It is possible to use a kit for diagnosis of oxaliplatin resistanceconsisting of:

a) at least one compartment suitable to contain and, as the case may be,containing a primer or a probe for a mitochondrial apoptosis gene suchas the Bax gene;

b) possibly the reagents necessary for the implementation of ahybridization reaction;

c) possibly at least one primer and the reagents necessary for a DNAamplification reaction.

According to another characteristic the invention relates to a processthat aims to determine if oxaliplatin treatment is to be pursued and/orcompleted, characterized in that it consists of:

a) obtaining at least two samples comprising cancer cells coming fromthe patient undergoing oxaliplatin treatment;

b) measurement of the level of mitochondrial apoptosis, for example bymeans of measurement of the expression of the Bax protein, in thesamples;

c) continuation of treatment when the level of apoptosis does notdecrease during treatment.

According to another characteristic, the invention relates to a processfor selection of compounds that inhibit oxaliplatin resistance,designated as anti-resistance compounds, the process consisting of themeasurement of the expression of at least one mitochondrial apoptosisgene before and after addition of a candidate compound to theoxaliplatin resistant cancer cells of a patient.

In vitro, the process can involve the addition of at least one candidatecompound to oxaliplatin resistant cancer cells sampled from a patient,the comparison of the level of mitochondrial apoptosis and/or expressionof apoptosis genes in the presence and absence of the compound, thededuction of the anti-resistance effect when the level of apoptosis isgreater after addition of the compound. The anti-resistance effect isalso deducted if the level of expression after addition of the compoundis greater when the gene is a gene that stimulates apoptosis, and lesserwhen the gene is a gene that is inhibitory of mitochondrial apoptosis.

In vivo the selection process may include, in a patient treated withoxaliplatin and resistant to oxaliplatin:

a) obtaining at first of a first sample consisting of cancer cells ofthe patient;

b) administration of the candidate compound to the patient;

c) obtaining later of a second sample consisting of cancer cells of thesame patient;

d) determination of the level of mitochondrial apoptosis and/or of thelevel of expression of at least a mitochondrial apoptosis gene such asthe Bax gene in the first or second sample;

e) deduction of the oxaliplatin anti-resistance effect of the compoundwhen the level of apoptosis is greater in the second sample.

The anti-resistance effect is also deducted if the level of expressionis greater in the second sample when the gene is a gene that stimulatesapoptosis, and lesser if the gene is a gene that inhibits mitochondrialapoptosis. Such in vivo procedures most preferably relate to compoundsderived from compounds already identified as anti-resistant.

By anti-resistance agent we mean a compound capable of reducing, mostpreferably of totally offsetting, the oxaliplatin resistance ofpatients. These anti-resistance agents are designed to restore thenormal level of expression of at least one mitochondrial apoptosis gene,either directly, or indirectly for example by activation or inhibitionof molecules regulating the expression of these genes. Ananti-resistance agent can for example block the activity of a compoundresponsible for abnormal inhibition of the activity of apoptosis genesat the level of transcription, translation, or activity of a protein.

Candidate compounds can be sought in particular among small molecules,polypeptides (for example oligopeptides, antibodies, antibody fragments)and nucleic acids. Targeting processes for oxaliplatin anti-resistanceagents typically involve banks of molecules known to those skilled inthe art such as banks of biological substances (in particular proteins)and banks of synthetic substances.

Banks of compounds can present themselves in solution form (e.g.,Houghten, 1992, Biotechniques 13:412-421), on beads (Lam, 1991, Nature354:82-84), on chips (Fodor, 1993, Nature 364:555-556). It is alsopossible to use banks described in the documents U.S. Pat. Nos.5,292,646 and 5,270,281.

It is possible to study in particular the effect of compounds alreadyknown to those skilled in the art as stimulators of mitochondrialapoptosis, such as TNF (Tumor Necrosis Factor), FasL, glutamate,Herbimycin A (Mancini et al., J. Cell. Biol. 138:449-469, 1997),Paraquat (Costantini et al., Toxicology 99:1-2, 1995), protein kinaseinhibitors such as Staurosporin, Calphostin C, derivatives ofd-erythro-sphingosine, Chelerythrine chloride, inducers of MAP kinasesuch as Anisomycin and inducers of the MPT category to which the Baxprotein belongs (Jurgenmeier et al., Proc. Natl. Acad. Sci. U.S.A.95:4997-5002, 1998).

Among the tests that make it possible to measure the level ofmitochondrial apoptosis, we may mention the measurement of the enzymaticactivity of the mitochondrial complexes ETC I, II, III, IV and of ATPsynthetase, the measurement of mitochondrial oxygen consumption (Milleret al., J. Neurochem., 67:1897, 1996), the measurement of the oxidationstate of mitochondrial cytochrome c at 540 nm and the measurement ofoxidative stress in the presence and absence of the anti-resistanceagent.

According to another characteristic the invention relates to the use ofat least one anti-resistance agent that stimulates mitochondrialapoptosis for the preparation of a medication in patients presenting orable to present resistance to oxaliplatin. By resistant patient we meana patient presenting cancer cells resistant to oxaliplatin. Such ananti-resistance agent can be used in patients presenting a partialresponse to oxaliplatin treatment in order to improve the efficacy ofthe treatment.

According to one realization the anti-resistance compounds are derivedfrom a selection process such as described previously. Those skilled inthe art have at their disposal tests sufficiently well-described in theapplication to select these compounds; the invention therefore alsocovers the use of these compounds, even if the precise chemicalstructure of the compounds is not completely identified: if a testedcompound fulfills the selection criteria (in particular stimulation ofapoptosis, increase in expression of at least one apoptosis stimulatinggene, reduction in expression of at least one apoptosis inhibitinggene), then those skilled in the art can use it for the preparation of amedication for anti-resistance to oxaliplatin without necessarilyneeding to know its chemical structure.

The treatment more specially targets cancer cells that have acquiredoxaliplatin resistance. The treatment aims to restore a level ofexpression or activity of the genes implicated in mitochondrialapoptosis that is sufficient so that the resistant cancer cells moreactively re-employ this process. Normal apoptosis is sought that issimilar to that of non-resistant cancer cells, or at least an increasein mitochondrial apoptosis sufficient to reduce clinical symptoms.

Treatment of the patient will typically involve the combination ofoxaliplatin and at least one anti-resistance agent, according to anadministration that can be simultaneous, separate or spaced out in time.The amount of the anti-resistance agents to be administered to thepatients must be sufficient to be therapeutically effective, in order toat least partially reduce oxaliplatin resistance. Treatment combiningoxaliplatin and at least one agent of anti-resistance to oxaliplatin, ina resistant patient, aims preferentially at obtaining a therapeuticefficacy at least equal to that of oxaliplatin treatment in anon-resistant patient.

The invention also relates to a method for treatment of anoxaliplatin-resistant patient or one capable of presenting oxaliplatinresistance, involving the administration of at least one compound thatstimulates mitochondrial apoptosis.

The invention also relates to a process for inhibiting oxaliplatinresistance in humans, involving the administration of a compound capableof selectively stimulating the mitochondrial apoptosis of cancer cells,in a patient requiring such an anti-resistance treatment.

The toxicity and therapeutic efficacy of anti-resistance agents can bedetermined by standard techniques of experimentation on cultured cellsor laboratory animals. Transposition to human patients knowing thesedata is obtained by means of appropriate methods.

A formulation according to the invention consists of oxaliplatintypically in the amount of 1 to approximately 10 mg/ml, most preferably1 to 5 mg/ml, and even more preferably from 2 to 5 mg/ml. Theoxaliplatin doses administered to the resistant patient will typicallybe on the order of 10 mg/m2/day to 250 mg/m2/day, preferably 20mg/m2/day to 200 mg/m2/day, most preferably between 50 and 150mg/m2/day.

Administration may be repeated for cycles of 1 to 5 days spaced apart byan interval of 1 to 5 weeks. For patients presenting strongerresistance, the clinician will determine the appropriate oxaliplatindose, the dose of anti-resistance agents and duration of treatment.

Should the occasion arise, the oxaliplatin and the anti-resistance agentcan be combined with at least one compound known to those skilled in theart to reinforce the efficacy and/or stability of the oxaliplatin; suchagents are described in the documents EP 0 943 331 and WO 01/66102.

The oxaliplatin will typically be combined with a pharmaceuticallyacceptable transporter, such as an appropriate solvent. The transporterwill in general be water, or one or more solvents, or a mixture of waterand one or more appropriate solvents. It might be preferred to use puresterile water for injection, and among solvents: polyalkylene glycolssuch as polyethylene glycol, polypropylene glycol, polybutylene glycoland analogues, ethanol, 1-vinyl-2-pyrrolidone polymer, solutions ofpharmaceutically acceptable sugars such as lactose, dextrose, sucrose,mannose, mannitol and cyclodextrins or analogues. The pH of theoxaliplatin solution formulations is typically 2 to 5, most preferablyfrom 3 to 4.5.

The formulations of this invention are to be administered to patients byappropriate conventional routes, typically by the parenteral route (forexample intravenous, intraperitoneal and analogues). Intravenousadministration is performed for example over a period of 12 hours to 5days. The percentage of the active compound in mixed formulationsaccording to the invention comprising oxaliplatin and at least oneresistance agent is adjusted according to the dosage and the degree ofresistance to oxaliplatin in particular. The appropriate dosage for aparticular patient is to be determined in particular as a function ofthe type of administration chosen, the duration of treatment, the size,the age, the physical condition of the patient, the degree ofoxaliplatin resistance and the response of the patient to thecomposition.

Incorporation of the anti-resistance agent in the oxaliplatincomposition is done by appropriate techniques.

For oral administration by means of pastilles, powders, granules andanalogues, it is possible to use excipients such as lactose, sodiumchloride, sucrose, glucose, urea, starch, calcium, kaolin, crystallinecellulose, salicylic acid, methyl cellulose, glycerol, sodium alginate,gum Arabic and analogues. It is possible to use usual binding agentssuch as glucose solutions, starch solutions and gelatin solutions. It ispossible to use disintegrants such as starch, sodium alginate, agarpowder and calcium carbonate. Among the absorbent agents, it is possibleto use starch, lactose, kaolin and bentonite. Among the lubricants, itis possible to use purified talc, salts of stearic acids andpolyethylene glycol.

Care will be taken in the formulation to avoid possible problems due tothe combination of oxaliplatin and an anti-resistance agent such asproblems with precipitation of the compounds.

A therapeutic oxaliplatin composition will typically contain 0.005% to95%, most preferably 0.5 to 50% oxaliplatin-ahd anti-resistance agents.

On the plane of mechanism of action, according to one realization, theanti-resistance agent is an agent that stimulates the expression of atleast one mitochondrial apoptosis gene.

According to one realization, the anti-resistance agent is a moleculecapable of inhibiting expression of genes that inhibit mitochondrialapoptosis. It is possible to use complementary anti-senseoligonucleotides of mRNA coding for molecules inhibiting the expressionof apoptosis effector genes. The anti-sense oligonucleotide will bindspecifically to the mRNA of such inhibitory molecules, inhibiting theirtranslation. The complementarity will have to be sufficient so thathybridization with the mRNA by the inhibitory molecule leads to theformation of stable hybrids. Typically, anti-sense strands will be usedwith a length between 6 and 50 nucleotides, typically of at least 10 to20 nucleotides. The anti-sense strands can be synthesized by methodsknown to those skilled in the art, using nucleotides modified toincrease the stability of the anti-sense/sense duplex. The followingmodified nucleotides can for example be used: 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, 5-methoxy-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil and 2,6-diaminopurine. Theanti-sense strands can also be produced biologically by means of avector for expression in which the anti-sense strand was sub-cloned inan anti-sense orientation.

Should the case arise, the anti-sense strand can be conjugated withpeptide molecules facilitating its transport or activity at the level ofthe targeted site of action. It is possible to inject anti-sensemolecules directly into a targeted area of the tissue and the anti-sensestrand may be linked to molecules such as peptides or antibodies capableof binding specifically to receptors expressed on the surface of thetarget cells.

Administration of anti-sense strands is to be such that these moleculescan act on an adequate level in the mitochondria.

According to another characteristic the invention relates to apharmaceutical composition consisting of oxaliplatin and at least oneanti-resistance agent capable of stimulating mitochondrial apoptosis, bystimulating expression of mitochondrial apoptosis genes or by blockingeffectors responsible for resistance.

According to one realization, the anti-resistance agent is an agent forregulation-stimulation of expression of the Bax gene, and/or an agentfor blocking of effectors of resistance.

Expression of mitochondrial apoptosis genes can be increased by transferof nucleic acids containing a sequence coding for the apoptosis geneand/or a regulatory sequence, by means of transfer techniquesappropriate for mitochondria. These sequences can be inserted inexpression vectors and transferred into the cells, for example by meansof plasmids. The nucleic acid inserted in the vector may code for thecomplete sequence of the apoptosis protein or a biologically activefragment with an activity most preferably of at least 50, 70, 90, 95% ofthe activity of the complete apoptosis protein.

The nucleic acids that can be used in the expression vectors can beoperationally linked to regulatory sequences such as a promoter orenhancer sequence that stimulates their expression. These regulatorysequences can be those naturally associated with the genes coding forapoptosis proteins.

Those skilled in the art are aware of a large number of appropriatetechniques for the transfer of nucleic acids into cells by vectorstypically plasmids, such as the liposome-polybren technique, DEAEdextran transfection (Felgner et al., Proc. Natl. Acad. Sci. USA,84:7413, 1987; Ono et al., Neurosci. Lett. 117:259, 1990; Brigham etal., Am. J. Med. Sci. 298:278, 1989), electropration (Neumann et al.,EMBO J., 7:841, 1980), precipitation with calcium phosphate (Graham etal., Virology, 52:456, 1973; Wigler et al., Cell, 14:725, 1978; Feigneret al., supra), microinjection (Wolff et al., Science, 247:1465, 1990)and biolistic techniques. Most preferably, vectors appropriate for atransfer of genes at the mitochondrial level are to be used, for exampleHBV virus (hepatitis B Virus), the transfer described for example in thedocument U.S. Pat. No. 6,100,068.

Thus, treatment of oxaliplatin resistance rests on the use of newtherapeutic processes, in particular resort to chemical substancesand/or gene therapies, capable of reducing resistance by restoringactivation of mitochondrial apoptosis normally caused by oxaliplatin inthe tumor cells of colorectal cancers.

This invention also has for object a cell HCT116/S as registered on 16Jun. 2003, under the number: I-3051, with the Collection Nationale deCultures de Microorganismes (CNCM), Pasteur Institute, Paris, France.

The line designated as HCT116/S comes from a sub-cloning of thewild-type line HCT116 of the ATCC (in order to ensure the clonality ofthese cells). This line HCT116/S was registered on 16 Jun. 2003, undernumber: I-3051, with the Collection Nationale de Cultures deMicroorganismes (CNCM) of the Pasteur Institute, Paris, France, underthe terms of the Budapest Treaty.

This invention also has for purpose the use of cell HCT116/S such asregistered with the CNCM on 16 Jun. 2003, under number: I-3051, or ofany cell derived from this HCT116/S cell, to study the correlationbetween the resistance of cancer cells, most preferably colorectal, toanti-cancer treatment and the expression and/or activity of amitochondrial apoptosis gene.

By cell derived from this HCT116/S cell as registered with the CNCM on16 Jun. 2003, under number: I-3051, we mean to designate here inparticular, any daughter cell originating from this line HCT116/S, orany variant cell originating from this line HCT116/S that has mostpreferably acquired resistance to a compound, such as an anti-cancercompound like oxaliplatin, or that has encountered a sensitivity to sucha compound (cf. for example cell lines originating from HCT116/Sdescribed below in the paragraph “Implementation of the ExperimentalModel”).

Such HCT116/S cells as those registered with the CNCM on 16 Jun. 2003,under number: I-3051, or their derivative cells can also be used for thevisualization and identification of a mitochondrial apoptosis gene whoseexpression is linked to the resistance of cancer cells, most preferablycolorectal, to an anti-cancer treatment, in particular to treatment withoxaliplatin.

Such HCT116/S cells as those registered with the CNCM on 16 Jun. 2003,under number: I-3051, or their derivative cells can also be used for theselection of a compound capable of stimulating mitochondrial apoptosisof a cancer cell, said compound being designed to be combined with ananti-cancer agent to which said cancer cell is resistant, mostpreferably said anti-cancer agent to which said cancer cell is resistantbeing oxaliplatin and, as the case may be, said cell is a colorectalcancer cell.

Such a process involves in particular a step in which said compound tobe tested and the anti-cancer agent to which said cancer cell isresistant are to be put together with said HCT116/S cells or theirderivative cells, followed by observation of the resistance of thesecells, in particular by studying the activity of mitochondrial apoptosisgenes, such as the activity of Bax and/or Bak, or also of genesimplicated in mitochondrial apoptosis such as cited above.

Other objects and advantages of the invention will appear upon readingof the detailed description that follows, illustrated by the followingfigures:

FIG. 1 shows that the HCT116R line, resistant to oxaliplatin, does notexpress the Bax protein;

FIGS. 2A to 2D show that the HCT116R and SW620R lines resist apoptoticinduction such as caused by oxaliplatin in the original HCT116 and SW620lines;

FIGS. 3A and 3B show that the HCT116R and SW620R lines also resistdirect mitochondrial apoptotic induction, which can be obtained underthe effect of the agents arsenic and lonidamine;

FIGS. 4A to 4C show that oxaliplatin sensitivity is associated with thedegree of activation of Bax;

FIGS. 5A to 5E show that oxaliplatin sensitivity is associated with thedegree of activation of Bak.

IMPLEMENTATION OF THE EXPERIMENTAL MODEL

Work was performed “in vitro” on colorectal cancer cell lines (CRC)obtained from the international collection managed in the United States(ATCC) and re-cloned in the laboratory. These lines are referenced, inparticular by the American Institute for Cancer Research (NCI/NIH), asstandards for pharmacological evaluation of anti-tumor drugs.

From these lines, sensitive to the cytotoxic effect of oxaliplatin, theinventors have isolated derivative lines capable of specificallyresisting oxaliplatin (and not the other medications cisplatin andirinotecan), by exposing these cells to increasing concentrations ofoxaliplatin, in a scheme suitable for the acquisition of resistance. Theresults presented in the application relate to the original lines,HCT116 and SW620, as well as their derivatives HCT116R (also designatedin the following as HCT116/R) and SW620R (also designated in thefollowing as SW620/R), respectively 70 and 20 times more resistant thanthe original lines.

Concerning the referenced line ATCC HCT116, two lines are thus describedin example 1 following:

the line designated HCT116, which is designated HCT116/S in example 2,originates from a sub-cloning of the wild-type line HCT116 of the ATCC(in order to ensure the clonality of these cells). This line HCT116/Swas the object of registration on 16 Jun. 2003, under number: I-3051, inthe Collection Nationale de Cultures de Microorganismes (CNCM) of thePasteur Institute, Paris, France, according to the provisions of theBudapest Treaty, in accordance with Rule 6.1;

the line designated HCT116R or HCT116/R, which is designated HCT116/R2in example 2, derived from the HCT116 line after acquisition ofoxaliplatin resistance and cloning (corresponds to the clone 70 timesmore resistant than the sensitive reference line HCT116/S).

In example 2, three other cellular variants are described. They are:

the line HCT116/R1 derived from the line HCT116 after acquisition ofoxaliplatin resistance and cloning (corresponding to a clone 30 timesmore resistant than the sensitive reference line HCT116/S),

the variant HCT116/Rev1 derived from the line HCT116/R1 after culture inthe absence of oxaliplatin for 6 months. This variant is characterizedby a return to the initial level of sensitivity (oxaliplatin sensitivitycomparable to HCT116/S),

the variant HCT116/Rev2 derived from the line HCT116/R2 after culture inthe absence of oxaliplatin for 15 months. This variant is characterizedby an only partial loss of oxaliplatin resistance (variant HCT116/Rev2is 16 times more resistant than line HCT116/S).

The lines HCT116/R1 and Rev1 do not carry the homozygous mutation of theBax gene identified in HCT116/R2 (monitored by sequencing of the regioncontaining codons 38 to 41). The level of expression of Bax isequivalent for HCT116/S, R1 and Rev1. The variant HCT116/Rev2 preservesthe homozygous mutation identified in HCT116/R2 as well as the absenceof Bax expression characteristic of this mutation.

EXAMPLE 1 Oxaliplatin Resistance in Lines HCT116R and SW620R Derivedfrom CRC Lines HCT116 and SW620, and Results

TABLE 1 “Lines HCT116R and SW620R derived from CRC lines HCT116 andSW620 are specifically resistant to oxaliplatin” IC₅₀(μM)^(a) Cell LineOxaliplatin Cisplatin Irinotecan HCT116 0.32 ± 0.08(1.0)^(b) 4.7 ± 1.8(1.0) 7.7 ± 3.8 (1.0) HCT116/R 21.9 ± 6.3 (68.4) 13.4 ± 6.6 (2.9) 9.5 ±4.2 (1.2) SW620  3.4 ± 0.6 (1.0) 7 ± 1.7 (1.0) 23.3 ± 0.6 (1.0) SW620/R62.3 ± 12.9(18.3) 9 ± 1.7 (1.4) 11.7 ± 2.5 (0.5)^(a)The inhibitory concentration 50 or IC is the concentration ofmedication that reduces cell growth by 50%. The values of IC₅₀ weremeasured by wustl colorimetric test after incubation of the medicationfor 48 hours. The values correspond to the average ± SD obtained from atleast three independent experiments.^(b)The numbers between parentheses correspond to relative resistance,determined by the ratio of the IC₅₀ of the resistant clone divided bythe IC₅₀ of the parental clone.

Table 1 shows that lines HCT116R and SW620R are approximately 70 timesand 20 times more resistant to oxaliplatin than the lines from whichthey are derived. They present very little or no crossed resistance tocisplatin or irinotecan. Their resistance is therefore specific foroxaliplatin.

The study was conducted at the same time on two cell models of differentgenetic backgrounds so as to be able to reinforce the significance ofthe observed results; thus, line SW620 originates from a metastasis andpossesses a mutated p53 regulatory protein whereas the HCT116 lineoriginates from an early tumor with microsatellite instability andpossesses a wild-type p53 protein. Observation of the alteration ofmitochondrial apoptosis associated with the resistant phenotype (seebelow), in two different cellular contexts, makes it possible togeneralize the results and therefore provide a high probability of itsmedical impact.

Exploration of the Experimental Model

The essential demonstrations were provided by these two distinct lines,so as to corroborate the universal character of the invention. Severalcomplementary studies were limited to the HCT116 line and its derivativeHCT116R.

The molecular mechanisms of oxaliplatin resistance of CRC cells beingunknown, the inventors did a comparison study on the genic expression ofthe sensitive and resistant phenotypes in the HCT116 model(transcriptome analysis). The inventors succeeded in identifying amarked reduction in the levels of messenger RNA of certain genes linkedto apoptosis, in particular of the Bax gene implicated in the pathreferred to as “Mitochondrial Apoptosis” (MA).

These observations were reinforced by biochemical analysis(immunoblotting indicates disappearance of Bax protein expression) andby sequencing of the Bax gene (in line HCT116R, a homozygous mutation ofthe Bax gene suppresses its expression).

FIG. 1:

FIG. 1 shows that the HCT116R line does not express the Bax protein,with or without oxaliplatin treatment, whereas the original HCT116 lineexpresses it without treatment and over-expresses it after oxaliplatintreatment. Sequencing showed that the HCT116R line is a homozygousmutant (deletion of a deoxyguanosine) in a region of the Bax genecontaining a series of 8 deoxyguanosines (codons 38 to 41), whichinterdicts its expression by shifting of the reading frame. The originalHCT116 line being heterozygous G8/G7, it therefore normally expressesthe Bax gene.

Legend to FIG. 1: detection of Bax by Western blot in the absence of, orunder the effect of treatment, with oxaliplatin in the HCT116 model. Thecells are not treated or treated with oxaliplatin at a level of 15 μMfor 48 hours (or 50 μM for 24 hours) before preparation of cell lysates.Tubulin expression is used as a control of equivalent protein deposits.

The inventors focused their work on the functional study of this path,in combination with oxaliplatin resistance. The principal resultsobtained are the following:

In the first place, the inventors showed that the lines HCT116R andSW620R, compared to the original lines, are resistant to induction ofapoptosis by oxaliplatin. The inventors also verified in the HCT116model that this resistance to apoptosis is specifically developed withrespect to oxaliplatin, since the HCT116R line remains sensitive toinduction of apoptosis by another anti-CRC medication (irinotecan) whosemechanism of action is different.

FIGS. 2A to 2D show that lines HCT116R and SW620R resist induction ofapoptosis by oxaliplatin. This resistance was specifically developedwith respect to oxaliplatin: the HCT116R line does not resist apoptoticinduction caused an anti-CRC medication with a different mode of action,irinotecan (cf. FIGS. 2A, 2B, 2D). Resistance to induction of apoptosisby oxaliplatin, observed by cytofluorometry after labeling by annexineV, is confirmed by lack of activation of caspase 3 (FIG. 2C).

Legend to FIGS. 2A to 2D: cells HCT116 (and R) and SW620 (and R) aretreated, for 48 hours prior to determination of the degree of apoptosis,by oxaliplatin (FIGS. 2A and 2B) or another anti-CRC medication,irinotecan, for cells HCT116 and HCT116R (FIG. 2D). A control isperformed without contact with any medication (Co). The degree ofapoptosis is then determined by cytofluorometry using labeling byannexine V. Independently, activation of the apoptosis effector proteinCaspase 3 was evaluated in cells HCT116 and HCT116R after treatment for24 hours with oxaliplatin in order to validate the entry into apoptosisof the cells as observed by cytofluorometry (FIG. 2C).

Subsequently, the inventors showed that the resistance to apoptosisinduced by oxaliplatin in lines HCT116R and SW602R is accompanied byresistance to induction of apoptosis by two chemical agents known to bedirect activators of MA (arsenic trioxide and lonidamine).

FIGS. 3A and 3B show that lines HCT116R and SW620R are resistant toapoptotic induction under the effect of the direct activators of MAarsenic and lonidamine.

Legend to FIGS. 3A and 3B: cells HCT116 (and R) and SW620 (and R) aretreated, prior to determination of the degree of apoptosis, for 24 hoursby arsenic trioxide (As), lonidamine (LND) or are left without treatment(control, Co). The degree of apoptosis is then determined bycytofluorometry after labeling with the dye “Mitocapture” whichfluoresces differently in apoptotic cells and intact cells (withrelation to mitochondrial integrity).

Thus the inventors showed that genetic, biochemical and functionalalterations of apoptosis are associated with oxaliplatin resistance.They relate to two CRC cell lines separately selected for their specificresistance to oxaliplatin. The relevance of this selection is validatedby the following characteristics:

The acquisition of oxaliplatin resistance is specific since it is notaccompanied by acquisition of cisplatin resistance (a molecule that isrelated and that very frequently presents crossed resistance tooxaliplatin) or irinotecan resistance (another molecule indicated in thetreatment of CRC as an alternative to oxaliplatin or in combination).

Oxaliplatin resistance, as well as functional alterations (resistance toapoptosis) is observed for an oxaliplatin concentration equivalent tothe plasma peak in man during treatments.

The inventors have shown that resistance to apoptosis is exerted at themitochondrial level. This is in particular shown by trials with directinducers of MA. These alterations are therefore diagnostic markers forthe resistance of colorectal cancers to oxaliplatin. Moreover, it isprobable that pharmacological modulation of the MA pathway will make itpossible to restore all or part of the sensitivity of CRCs tooxaliplatin. The inventors have verified, by a several months follow-upstudy of the derivative cell lines, that their resistance phenotype isspontaneously reversible in the absence of pharmacological pressure(=cell culture without oxaliplatin). This reversibility, total orpartial according to the case, makes it possible to predict reversionunder the effect of a substance opposing or circumventing the mechanismsof resistance within MA.

The inventors have moreover developed the purification of mitochondriafrom sensitive and resistant lines in order to isolate and test putativeeffectors of resistance (like PTPC), as well as agents blocking theseeffectors (anti-sense RNA, substances already known to block aphysiological mechanism at the level of MA, etc.). The invention alsocovers the implementation of gene transfers restoring the phenotype ofoxaliplatin sensitivity, and of processes for targeting of new chemicalentities that make it possible to oppose or circumvent resistance, fromeffectors as targets and from banks of chemical substances as sources.

EXAMPLE 2 Activation of Bax and Bak

Activation of Bax

Bax is present in the cells in two forms: a latent (inactive) form andan active form that participates in the apoptotic process.

The overall level of Bax expression is equivalent for the linesHCT116/S, HCT116/R1 and HCT116/Rev1. Exposure to oxaliplatin induces anover-expression of Bax. This over-expression remaining comparable overall these lines, the inventors have tried to find out if the degree ofBax activation could account for the response of cells to oxaliplatin orif Bax definitely, taken by itself, couldn't be systematicallyassociated with oxaliplatin sensitivity.

FIGS. 4A to 4C: Sensitivity to oxaliplatin associated with degree of Baxactivation.

Legend to FIGS. 4A to 4C: Detection of activation of Bax by an antibodyspecific for the active conformation of Bax and intracellular analysisby flow cytometry. The cells are fixed, permeabilized and then incubatedwith the antibody specific for the active conformation of Bax. Finallyvisualization is carried out by incubation with the secondary antibodycoupled with FITC. The labeling of untreated cells is shown by the curvein fine black line. That of treated cells (12, 24 or 48 hours with 15 μMoxaliplatin) is shown by the curves in thick black line. The appearanceof strongly labeled cells (curves in thick black line shifted to theright) gives evidence of activation of Bax. The experiment wasreproduced three times and comparable results were obtained.

The inventors have demonstrated by means of FIGS. 4A to 4C that thestate of resistance or sensitivity of cells to oxaliplatin correlateswell with the degree of activation of Bax. The activation is reduced anddelayed in the resistant line HCT116/R1 (FIG. 4B) compared to thesensitive line HCT116/S (FIG. 4A). Return to the state of sensitivity ofthe revertant HCT116/Rev1 (FIG. 4C) is accompanied by return to earlyactivation of Bax.

Activation of Bak

The pro-apoptotic molecule Bak plays a preponderant role in the same wayas Bax in apoptosis mediated mitochondrial permeabilization. These twomolecules seem to have functions that are very closely related andsometimes redundant. The inventors have therefore also tried to find outif there is a relationship between the level of activation of Bak and aresponse to oxaliplatin over all lines including the lines HCT116/R2 andRev2 mutated in Bax.

FIGS. 5A to 5E: Oxaliplatin sensitivity associated with the degree ofactivation of Bak.

Legend to FIGS. 5A to 5E: Detection of activation of Bak by an antibodyspecific for the active conformation of Bak and intracellular analysisby flow cytometry. The cells are fixed, made permeable and thenincubated with the antibody specific for the active conformation of Bax.Finally visualization is carried out by incubation with the secondaryantibody coupled to FITC. Labeling of untreated cells is shown by thecurve in the fine black line. That of treated cells (12, 24 or 48 hourswith 15 μM oxaliplatin) is shown by the curves in the thick black line.The appearance of strongly labeled cells (curves in the thick black lineshifted to the right) gives evidence of activation of Bak. Thisexperiment was reproduced two times.

The inventors have demonstrated by means of FIGS. 5A to 5E that theactivation of Bak in response to oxaliplatin treatment is delayed in theresistant lines HCT116/R1 (FIG. 5B) and HCT116/R2 (FIG. 5C). Therevertants Rev1 (FIG. 5D) and Rev2 (FIG. 5E) repeat the level ofactivation of Bak of the sensitive line HCT116/S (FIG. 5A). As is thecase with Bax, activation of Bak correlates well with the response ofthe cells to oxaliplatin.

In conclusion, a co-activation of Bax and Bak can be observed inresponse to oxaliplatin. Early activation of Bax and Bak is associatedwith the state of sensitivity of the cells to oxaliplatin.

1. Process for in vitro detection of resistance of cancer cells tooxaliplatin treatment, characterized in that it involves the measurementof the mitochondrial apoptosis of cancer cells that are treated or canor are to be treated with oxaliplatin.
 2. Process according to claim 1,characterized in that the cancer is a cancer treated with oxaliplatin,in particular a colorectal cancer, a cancer of the ovaries, a cancer ofthe germinal cells, a cancer of the lung, a cancer of the digestivetract, a cancer of the prostate, a cancer of the pancreas, a cancer ofthe small intestine or a cancer of the stomach.
 3. Process according toclaim 1 or 2, characterized in that it involves the measurement of theexpression of at least one mitochondrial apoptosis gene.
 4. Processaccording to any of claims 1 to 3, characterized in that it involves themeasurement of the expression of at least one gene coding for a Bax,Bcl-2 or cytochrome c protein.
 5. Process according to claim 3 or 4,characterized in that it involves the measurement of mRNA transcripts ofthe mitochondrial apoptosis genes.
 6. Process according to claim 3 or 4,characterized in that it involves measurement of the amount and/oractivity of mitochondrial apoptosis proteins in the cancer cells. 7.Process for in vitro detection of the resistance of cancer cells tooxaliplatin treatment characterized in that it involves the detection ofat least one mutation indicative of deficient mitochondrial apoptosis inthe case of treatment with oxaliplatin, in particular of a mutation in aregion of the Bax gene containing a series of 8 deoxyguanines. 8.Process according to any of claims 1 to 6, characterized in that itinvolves: a) determination of the level of mitochondrial apoptosis,and/or the level of expression of at least one mitochondrial apoptosisgene, in cancer cells sampled from a patient; b) comparison to the levelmeasured with a control sample of cells not resistant to oxaliplatin. 9.Process according to claim 6, characterized in that it involves puttingcancer cells together with an antibody capable of recognizing amitochondrial apoptosis protein or a biologically active fragment, andthe visualization of the antigen-antibody complex possibly formed. 10.Process according to any of claims 1 to 5, characterized in that itimplements a primer or probe sequence specific for the mitochondrialapoptosis gene.
 11. Process according to claim 10, characterized in thatit involves: a) possible isolation of mitochondrial DNA from thebiological sample to be examined, or the obtaining of a cDNA from theRNA of the biological sample or from genomic DNA; b) specificamplification of the DNA from a) by means of at least one primer foramplification of the mitochondrial apoptosis gene.
 12. Process accordingto claim 10, characterized in that it involves: a) putting a nucleotideprobe of an apoptosis gene together with the biological sample analyzed,the nucleic acid of the sample having, if need be, been previously madeaccessible to hybridization, under conditions allowing hybridization ofthe probe and the nucleic acid of the sample; b) visualization of thehybrid possibly formed.
 13. Process for selection of compounds thatinhibit the resistance of cancer cells to oxaliplatin, characterized inthat it involves: a) addition of at least one candidate compound to thecancer cells resistant to oxaliplatin; b) comparison of the level ofmitochondrial apoptosis and/or expression of at least one apoptosis genein the presence and absence of the compound; c) deduction of theanti-resistance effect when the level of mitochondrial apoptosis isgreater after addition of the compound, or when the level of expressionis greater when the gene is a gene that stimulates mitochondrialapoptosis, or when the level of expression is less when the gene is agene that inhibits mitochondrial apoptosis.
 14. Use of at least oneagent stimulating mitochondrial apoptosis, in particular chosen fromamong TNF, FasL, glutamate, Herbimycin A, Paraquat, inhibitors ofprotein kinase such as Staurosporine, Calphostin C, derivatives ofd-erythro-sphingosine, Chelerythrine chloride, inducers of MAP kinasesuch as Anisomycin and inducers of MPT for the preparation of amedication for patients presenting or capable of presenting oxaliplatinresistance.
 15. Use according to claim 14 for the preparation of amedication against colorectal cancer, or cancer of the ovaries, thegerminal cells, the lung, the digestive tract, the prostate, thepancreas, the small intestine or the stomach.
 16. Use according to claim14 for the preparation of a medication against colorectal cancers. 17.Product containing oxaliplatin and an agent stimulating mitochondrialapoptosis, in particular chosen from among TNF, FasL, glutamate,Herbimycin A, Paraquat, inhibitors of protein kinase such asStaurosporine, Calphostin C, derivatives of d-erythro-sphingosine,Chelerythrine chloride, inducers of MAP kinase such as Anisomycin andinducers of MPT as a combination product for simultaneous use, separatedor spaced apart in time as an anti-cancer agent.
 18. Compositionconsisting of oxaliplatin and at least one anti-resistance agent capableof stimulating mitochondrial apoptosis, chosen from among TNF, FasL,glutamate, Herbimycin A, Paraquat, inhibitors of protein kinase such asStaurosporine, Calphostin C, derivatives of d-erythro-sphingosine,Chelerythrine chloride, inducers of MAP kinase such as Anisomycin andinducers of MPT.
 19. Kit for diagnosis of resistance of a cancer tooxaliplatin characterized in that it includes: a) at least onecompartment suitable to contain a probe; b) possibly the reagentsnecessary for the implementation of a hybridization reaction; c)possibly at least one primer and the reagents necessary for a DNAamplification reaction.
 20. Cell HCT116/S as registered on 16 Jun. 2003,under number: I-3051, with the Collection Nationale de Cultures deMicroorganismes (CNCM), Pasteur Institute, Paris, France.
 21. Use ofcell HCT116/S according to claim 20, or of any cell derived from thiscell HCT116/S, to study the correlation between the resistance of cancercells, most preferably colorectal, to anti-cancer treatment and theexpression of a mitochondrial apoptosis gene.
 22. Use of cell HCT116/Saccording to claim 20, or of any cell derived from this cell HCT116/S,for the visualization and identification of a mitochondrial apoptosisgene whose expression is linked to the resistance of cancer cells, mostpreferably colorectal, to anti-cancer treatment.
 23. Use of cellHCT116/S according to claim 20, or of any cell derived from this cellHCT116/S, for the selection of a compound capable of stimulatingmitochondrial apoptosis in a cancer cell, said compound being designedto be combined with an anti-cancer agent to which said cancer cell isresistant, most preferably said anti-cancer agent to which said cancercell is resistant being oxaliplatin and, as the case may be, said cellis a colorectal cancer cell.